Homologous gene replacement offers a powerful method for altering and testing gene function (Capecchi, M. (1989) Science 244:1288-1292). In diploid organisms with a sexual cycle, a single heterozygous replacement is first obtained which is then rendered homozygous by sexual crossing. Although Leishmania are diploid at most loci, they appear to be predominantly or exclusively asexual in nature and in the laboratory (Tait, A. (1983) Parasitology 86:29-57; Tibayrenc, M. et al. (1990) Proc. Natl. Acad. Sci. USA 87:2414-2418; Panton, L. J. et al. (1991) J Protozoology 38:224-228). This situation is not unusual, as many diploid unicellular organisms lack a sexual cycle (Whelan, W. L. (1987) CRC Critical Reviews 14:99-170) or possess one that is not readily manipulable.
Methods have been developed that can be utilized to obtain homozygous mutant lines from a heterozygous parent, in the absence of a manipulable sexual cycle. Parasexual crossing has been utilized in some organisms; however, parasex has not been demonstrated in organisms such as Leishmania. Another approach is the use of UV radiation or other agents to induce mitotic recombination following transformation, as shown in Candida albicans (Kelley, S. L. et al. (1988) Science 241:1813-1815). The disadvantage is that mutagenic agents may create secondary mutations that cannot be removed by back-crossing.
Improved methods of homozygous gene replacement in diploid organisms which lack a sexual cycle are needed.